The present invention is generally related to substituted piperidine compounds and more particularly to compounds with activity as NMDA receptor subtype selective blockers that have low activity as blockers of hERG potassium channels.
The present invention relates to the compound of formula 
to its R,R- and S,S-enantiomers and to their pharmaceutically acceptable acid addition salts.
The compounds of the present invention are NMDA (N-methyl-D-aspartate)-receptor-subtype selective blockers, which have a key function in modulating neuronal activity and plasticity which makes them key players in mediating processes underlying development of CNS including learning and memory formation and function.
Under pathological conditions of acute and chronic forms of neurodegeneration overactivation of NMDA receptors is a key event for triggering neuronal cell death. NMDA receptors are composed of members from two subunit families, namely NR-1 (8 different splice variants) and NR-2 (A to D) originating from different genes. Members from the two sub-unit families show a distinct distribution in different brain areas. Heteromeric combinations of NR-1 members with different NR-2 sub-units result in NMDA receptors, displaying different pharmacological properties. Possible therapeutic indications for NMDA receptor subtype specific blockers include acute forms of neurodegeneration caused, e.g., by stroke or brain trauma; chronic forms of neurodegeneration such as Alzheimer""s disease, Parkinson""s disease, Huntington""s disease or ALS (amyotrophic lateral sclerosis); neurodegeneration associated with bacterial or viral infections, diseases such as schizophrenia, anxiety and depression and acute/chronic pain.
Objects of the present invention are novel compounds of formula I, its R,R- and S,S-enantiomers, racemic mixtures of these enantiomers and pharmaceutically acceptable salts of these novel compounds; their use in the treatment or prophylaxis of diseases caused by overactivation of respective NMDA receptor subtypes, which include acute forms of neurodegeneration caused, e.g., by stroke or brain trauma; chronic forms of neurodegeneration such as Alzheimer""s disease, Parkinson""s disease, Huntington""s disease or ALS (amyotrophic lateral sclerosis); neurodegeneration associated with bacterial or viral infections, and diseases such as schizophrenia, anxiety, depression and acute/chronic pain; the use of these compounds for manufacture of corresponding medicaments; processes for the manufacture of these novel compounds; and medicaments containing the compounds of the invention.
The term xe2x80x9cpharmaceutically acceptable acid addition saltsxe2x80x9d embraces salts with inorganic and organic acids, such as hydrochloric acid, nitric acid, sulfuric acid, lactic acid, phosphoric acid, citric acid, formic acid, fumaric acid, maleic acid, acetic acid, succinic acid, tartaric acid, methane-sulfonic acid, p-toluenesulfonic acid and the like.
4-Hydroxy-piperidin derivatives are described, for example in EP 824 098, in which the piperidine ring is substituted by one hydroxy group in 4-position. These compounds are described to possess activities on the NMDA receptor and are useful in the treatment of acute forms of neurodegeneration caused, for example, by stroke and brain trauma, and chronic forms of neurodegeneration such as Alzheimer""s disease, Parkinson""s disease, ALS (amyotrophic lateral sclerosis), neurodegeneration associated with bacterial or viral infections and acute/chronic pain.
It is known from EP 824 098 that these compounds are good NMDA receptor subtype specific blockers with a high affinity for NR2B subunit containing receptors and low affinity for NR2A subunit containing receptors.
Activity versus xcex11-adrenergic receptors is also low and the compounds are active in vivo against audiogenic seizures in mice in the low mg/kg range. Importantly, these compounds were neuroprotective in an animal stroke model, namely, a permanent occlusion of the middle cerebral artery. However, in vitro and in vivo cardiotoxicity studies showed that these compounds had the propensity to prolong cardiac action potential duration in vitro and consequently the xe2x80x98QTxe2x80x99-interval in vivo and thus, had a potential liability to produce cardiac arrhythmias. The ability of such compounds to prolong the cardiac action potential was identified as being due to an action at the hERG type potassium channel, which is important for action potential repolarisation in humans and other species, and most compounds known to prolong the QT-interval in man are active at blocking this channel. Thus, the compounds of the prior art block heterologously recombinant human ERG potassium channels.
It has now surprisingly been found that the following preferred compounds of formula I (3R,4R) and (3S,4S)-4-benzyl-1-[2-(4-hydroxy-phenoxy)-ethyl]-piperidine-3,4-diol, (3R,4R)-4-benzyl-1-[2-(4-hydroxy-phenoxy)-ethyl]-piperidine-3,4-diol and (3S,4S)-4-benzyl-1-[2-(4-hydroxy-phenoxy)-ethyl]-piperidine-3,4-diolare NMDA NR2B subtype selective antagonists. These preferred compounds of the invention share the highly specific subtype selective blocking properties of compounds of the prior art, for example of 1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol (EP 824 098), and are neuroprotectants in vivo, unlike the compounds of EP 824098, the preferred compounds of the invention are significantly less active as blockers of the hERG potassium channels and, thus, are much less likely to have pro-arrhythmic activity in man.
In the following table the high selectivity of compounds of the present invention is demonstrated.
The novel compounds of formula I and their pharmaceutically acceptable salts can be prepared by methods known in the art, for example by a process described below, which process comprises
reacting a compound of formula 
with a compound of formula 
and deprotecting the hydroxy group to give compounds of formulae 
and, preferably, converting the compounds obtained into a pharmaceutically acceptable acid addition salts.
In accordance with the described process variant, 4-benzyl-3,4-dihydroxy-piperidine, (3R,4R)-4-benzyl-3,4-dihydroxy-piperidine or (3S,4S)-4-benzyl-3,4-dihydroxy-piperidine is treated with 1-benzyloxy-4-(2-chloro-ethoxy)-benzene in the presence of K2CO3. The reaction is carried out at about 80-100xc2x0 C. The O-protecting group is then cleaved off in conventional manner, for example by hydrogenating in the presence of Pd/C.
The acid addition salts of the compounds of formula I are especially well suited for a pharmaceutical use.
The following schemes 1 and 2 describe the preparation of the compound of formula I and its desired enantiomeric forms. The starting materials of formulae III and 1-benzyloxy-4-(2-chloro-ethoxy)-benzene are known compounds or can be prepared by methods known in the art.
In schemes 1 and 2 the following abbreviations have been used:

wherein xe2x80x9chalxe2x80x9d may be chloro or bromo. 
The detailed description of the above mentioned processes is described in Examples 1-17.
As mentioned earlier, the compounds of formula I and their pharmaceutically acceptable addition salts possess valuable pharmacodynamic properties. They are NMDA-receptor subtype selective blockers, which have a key function in modulating neuronal activity and plasticity which makes them key players in mediating processes underlying development of CNS as well as learning and memory formation.
The compounds were investigated in accordance with the test given hereinafter.
Male Fxc3xcllinsdorf albino rats weighing between 150-200 g were used. Membranes were prepared by homogenization of the whole brain minus cerebellum and medulla oblongata with a Polytron (10,000 rpm, 30 seconds), in 25 volumes of a cold Tris-HCl 50 mM, EDTA 10 mM, pH 7.1 buffer. The homogenate was centrifuged at 48.000 g for 10 minutes at 4xc2x0 C. The pellet was resuspended using the Polytron in the same volume of buffer and the homogenate was incubated at 37xc2x0 C. for 10 minutes. After centrifugation the pellet was homogenized in the same buffer and frozen at xe2x88x9280xc2x0 C. for at least 16 hours but not more than 10 days. For the binding assay the homogenate was thawed at 37xc2x0 C., centrifuged and the pellet was washed three times as above in a Tris-HCl 5 mM, pH 7.4 cold buffer. The final pellet was resuspended in the same buffer and used at a final concentration of 200 xcexcg of protein/ml.
[3H]-Ro 25-6981 binding experiments were performed using a Tris-HCl 50 mM, pH 7.4 buffer. For displacement experiments 5 nM of 3H-Ro 25-6981 were used and non specific binding was measured using 10 xcexcM of tetrahydroisoquinoline and usually it accounts for 10% of the total. The incubation time was 2 hours at 4xc2x0 C. and the assay was stopped by filtration on Whatman GF/B glass fiber filters (Unifilter-96, Packard, Zxc3xcrich, Switzerland). The filters were washed 5 times with cold buffer. The radioactivity on the filter was counted on a Packard Top-count microplate scintillation counter after addition of 40 mL of microscint 40 (Canberra Packard S.A., Zxc3xcrich, Switzerland).
The effects of compounds were measured using a minimum of 8 concentrations and repeated at least once. The pooled normalized values were analyzed using a non-linear regression calculation program which provide IC50 with their relative upper and lower 95% confidence limits (RS1, BBN, USA).
Male Fxc3xcllinsdorf albino rats weighing between 150-200 g were used. Membranes were prepared by homogenization of the whole brain minus cerebellum and medulla oblongata with a Plytron (10,000 rpm, 30 seconds), in 25 volumes of a cold Tris-HCl 50 mM, EDTA 10 mM, pH 7.1 buffer. The homogenate was centrifuged at 48.000 g for 10 minutes at 4xc2x0 C. The pellet was resuspended using the Polytron in the same volume of buffer and the homogenate was incubated at 37xc2x0 C. for 10 minutes. After centrifugation the pellet was homogenized in the same buffer and frozen at xe2x88x9280xc2x0 C. for at least 16 hours but not more than 10 days. For the binding assay the homogenate was thawed at 37xc2x0 C., centrifuged and the pellet was washed three times as above in a Tris-HCl 5 mM, pH 7.4 cold buffer. The final pellet was resuspended in the same buffer and used at a final concentration of 200 mg of protein/ml.
3H-Prazosin binding experiments were performed using a Tris-HCl 50 mM, pH 7.4 buffer. For displacement experiments 0.2 nM of 3H-Prazosine were used and non specific binding was measured using 100 mM of Chlorpromazine. The incubation time was 30 minutes at room temperature and the assay was stopped by filtration on Whatman GF/B glass fiber filters (Unifilter-96, Canberra Packard S.A., Zxc3xcrich, Switzerland). The filters were washed 5 times with cold buffer. The radioactivity on the filter was counted on a Packard Top-count microplate scintillation counter after addition of 40 ml of microscint 40 (Canberra Packard S.A., Zxc3xcrich, Switzerland). The effects of compounds were measured using a minimum of 8 concentrations and repeated at least once. The pooled normalized values were analyzed using a non-linear regression calculation program which provide IC50 with their relative upper and lower 95% confidence limits (RS1, BBN, USA).
The thus-determined activity of compounds in accordance with the invention is in the range of 0.039-0.045 (in xcexcM), as described in the table above.
CHO cells were stably transfected by a pcDNA3-hERG expression vector containing a SV40-neo cassette for selection. Cells were thinly plated into 35 mm dishes and used for the electrophysiological experiment xc2xd-3 d later.
During the experiment the cells were continuously superfused with an extracellular saline containing (in mM): NaCl 150, KCl 10, MgCl2 1, CaCl2 3, HEPES 10 (pH=7.3 by addition of NaOH). A 10-mM stock solution of the test compound was made from pure DMSO. Test solution were made by at least 1000-fold dilution of the stock solution into the extracellular saline. The glass micropipettes for whole-cell patch-clamp recording were filled with a containing (in mM): KCl 110, BAPTA 10, HEPES 10, MgCl2 4.5, Na2ATP 4, Na2-phosphocreatine 20, creatine kinase 200 xcexcg/ml (pH=7.3 by addition of KOH).
The whole-cell configuration of the patch-clamp technique was used for the experiments. Cells were clamped to xe2x88x9280 mV holding potential and repetitively (0.1 Hz) stimulated by a voltage pulse pattern consisting of a 1-s conditioning depolarisation to 20 mV immediately followed by a hyperpolarisation of 50 ms duration to xe2x88x92120 mV. The membrane current was recorded for at least 3 min (18 stimuli) before compound application (control), and then for another two 3-min intervals in presence of two different concentrations of the compound. The current amplitudes (Itest) at the end of each compound application interval were divided by the mean current amplitude (Icontrol) during the initial control period to calculate the percentage effect of the compound:
effect (%)=(1-Itest/Icontrol)xc2x7100. 
Compound concentrations were chosen in decade steps (usually 1 and 10 xcexcM) around the expected 50% inhibitory concentration (IC50). If after the first experiment the IC50 turned out to lie outside the range between the two chosen concentrations the concentrations were changed to bracket the IC50 in the following experiments. The compound was tested on at least three cells. Its IC50 was then estimated from the population of all percent-effect values by non-linear regression using the function effect=100/(1xe2x88x92IC50/concentration)Hill). Concentrations higher than 10 xcexcM were not tested. If 10 xcexcM of the compound turned out to produce less than 50% effect, IC50 was labelled as xe2x80x9c greater than 10 xcexcMxe2x80x9d and the compound was characterised by the average effect seen at 10 xcexcM.
The compounds of formula I and their salts, as herein described, together with pharmaceutically inert excipients are preferably incorporated into standard pharmaceutical dosage forms, for example, for oral or parenteral application with the usual pharmaceutical adjuvant materials, for example, organic or inorganic inert carrier materials, such as, water, gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, gums, polyalkylene-glycols and the like. Examples of pharmaceutical preparations in solid form are tablets, suppositories, capsules, or in liquid form are solutions, suspensions or emulsions. Pharmaceutical adjuvant materials include preservatives, stabilizers, wetting or emulsifying agents, salts to change the osmotic pressure or to act as buffers. The pharmaceutical preparations can also contain other therapeutically active substances.
The daily dose of compounds of formula I to be administered varies with the particular compound employed, the chosen route of administration and the recipient. Representative of a method for administering the compounds of formula I is by the oral and parenteral type administration route. An oral formulation of a compound of formula I is preferably administered to an adult at a dose in the range of 1 mg to 1000 mg per day. A parenteral formulation of a compound of formula I is preferably administered to an adult at a dose in the range of from 5 to 500 mg per day.
The invention is further illustrated in the following examples.